2.3. Viral RNA quantification

For AIV, quantitative real time RT-PCR (qRRT-PCR) was performed as previously described (^{Kapczynski et al., 2013, Spackman et al., 2002}). Briefly, qRRT-PCR reactions targeting the influenza virus M gene was conducted using AgPath-ID one-step RT-PCR Kit (Ambion, Austin, TX) and the ABI 7500 Fast Real-Time PCR system (Applied Biosystem, Calsbad, CA). The RT step conditions for reactions were 10 min at 45 °C and 95 °C for 10 min. The cycling conditions were 45 cycles of 15 s, 95 °C; 45 s, 60 °C. For virus quantification, a standard curve was established with RNA extracted from dilutions of the same titrated stock of the viruses. Ct (cycle threshold) values of each viral dilution were plotted against viral titers. The resulting standard curve had a high correlation coefficient (r² > 0.99), and it was used to convert Ct values to EID₅₀/ml.

For NDV, qRRT-PCR targeting the NDV M gene was conducted as described previously (^{Wise et al., 2004}). The RT step was 30 min at 50 °C, followed by 15 min at 95 °C. The cycling conditions consisted of 40 cycles of 10 s of denaturation at 94 °C, 30 s of annealing at 52 °C, and extension at 72 °C for 10 s. For virus quantification, a standard curve was established with RNA extracted from dilutions of the titrated stock of the virus qRRT-PCR was conducted using AgPath-ID one-step RT-PCR Kit (Ambion) and the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Calsbad, CA).

For IBV, qRRT-PCR for IBV detection was performed as described previously (^{Callison et al., 2006}). The reaction was conducted at 50 °C for 30 min; 95 °C for 15 min; 40 cycles of 94 °C for 1 s followed by 60 °C for 60 s. For virus quantification, a standard curve was established with RNA extracted from dilutions of the titrated stock of the virus. RRT-PCR was conducted using AgPath-ID one-step RT-PCR Kit (Ambion) and the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Calsbad, CA).